One major area of investigation of our lab has been defining inter-organelle association and communication. In one project, we presented a systems-level analysis of the organelle interactome using a multispectral image acquisition method that overcomes the challenge of spectral overlap in the fluorescent protein palette. We used confocal and lattice light sheet instrumentation and an imaging informatics pipeline of five steps to achieve mapping of organelle numbers, volumes, speeds, positions and dynamic inter-organelle contacts in live cells from a monkey fibroblast cell line. We described the frequency and locality of two-, three-, four- and five-way interactions among six different membrane-bound organelles (endoplasmic reticulum, Golgi, lysosome, peroxisome, mitochondria and lipid droplet) and showed how these relationships change over time. We demonstrated that each organelle has a characteristic distribution and dispersion pattern in three-dimensional space and that there is a reproducible pattern of contacts among the six organelles, that is affected by microtubule and cell nutrient status. This methodology offers a powerful descriptive tool and can be used to develop hypotheses about cellular organization and dynamics. In another project, we developed a model of endosomal membrane sorting using macropinocytic cells. Macropinocytosis is an actin driven form of endocytosis that generates enlarged endosomes that can be unambiguously tracked using live-cell microscopy. Upon formation and internalization into the cell interior, membrane and proteins on the limiting membrane of the endosome can be transported to other organelles such as Golgi or lysosomes, or recycled back to the cell surface. Due to the large size of nascent endosomes, macropinocytic cells provide a unique model system to study sorting machinery and trafficking pathways with increased resolution. We have been able to resolve protein-specific, sorting events on individual endosomes, as well as observe recruitment of retromer machinery to isolated endosomal domains. We have employed a variety of microscopy techniques in these studies, including correlative light/electron microscopy (CLEM), live cell Airyscan super-resolution microscopy, photoactivation and photobleaching. We are further developing fluorescent probes to track protein delivery from the endosome to specific organelle compartments with the goal of better visualizing inter-organelle trafficking. A third project has focused on studying the ability of the Hippo signaling effector, YAP/TAZ, to sense and respond to cellular signals such as cytosolic volume changes. Mouse embryonic stem cells can grow in discrete pluiropotency states (nave and primed) which display differences in cell volume, shape, and confinement in cell culture. Nave ESCs grow clumped together in spheroid colonies, while primed ESCs flatten out into a more traditional monolayer. In comparing the two distinct pluripotency states of ESCs we demonstrated not only differences in baseline metabolism, with primed ESCs mainly depending on glycolysis and nave ESCs utilizing both glycolysis and oxidative phosphorylation, but also a differential YAP/TAZ nuclear localization in these two cell states. To understand if the YAP/TAZ localization could be a response to volumetric differences in the cell states, we tested YAP/TAZ localization in response to cytosolic volume reduction induced by hyperosmotic shock in multiple cell lines. Not only did YAP/TAZ increase its nuclear localization during hyperosmotic shock, but we observed its localization to phase-separated liquid droplets dependent upon the YAP1 transcription activation domain. We are further characterizing the effects of downstream signaling due to YAP/TAZ accumulation in liquid droplets. In a fourth project, we have been characterizing the relationship between calcium signaling and vesicular movements in cultured neuronal cells. Local calcium spikes have been reported to inhibit the movement of mitochondria in cultured neurons, which is important for neuronal activity-dependent recruitment of mitochondria to distinct locations. Using live-cell confocal imaging in astrocytes and neurons overexpressing the calcium sensor GCaMP6f, we further demonstrated the ability of local calcium spikes to inhibit movement of endososomes and lysosomes as well, indicating a broader role for calcium signals in organizing localization of multiple organelles in neurons. We plan to further explore the broader role of calcium-mediated organelle recruitment during brain organoid differentiation and development from iPSC cell lines.